The present invention relates to mesomorphic complexes of vitamin A acid and cationic polyelectrolytes, in particular in the form of films or nanodispersions, to process for their preparation and to the use of the mesomorphic complexes as vitamin A substitute.
Vitamin A acid is a highly crystalline low molecular weight material. Lipophilic hormones such as vitamin A acid, steroids, thyroid hormones and vitamin D3 act by binding to ligand-activated transcription factors comprising the steroid/nuclear receptor superfamily (R. M. Evans, Science 240 (1988), 889). Intensive investigations are currently in progress into the role of vitamin A acid in cell differentiation by investigating the binding properties of the retinoids to specific proteins (W. Bourguet, M. Ruff, P. Chambon, H. Gonemeyer and D. Moras, Nature 375 (1995), 377; J. -P. Renaud, N. Rochel, M. Ruff, V. Vivat, P. Chambon, H. Gronemeyer and D. Moras, Nature 378 (1995), 681).
In addition to their important role in the transmission of pleitrophic effects on morphogenesis, differentiation and hemostasis during the embryonic and postnatal phase of life, vitamin A acid shows a great potential as pharmacological active substance. At present, vitamin A acid is used for the external treatment of severe cases of acne, and its use for courses of skin rejuvenation has also been suggested (A. H. Lewin, M. E. Bos, F. C. Zusi, X. Nair, G. Whiting, Bouquin, G. Tetrault and F. I. Carroll, Pharm. Res 11 (1994), 192). Finally, there is also evidence of an inhibition of malignant tumors by retinoids (G. Zanotti, M. R. D""Acunto, G. Malpeli, C. Folli and R. Berni, Eur. J. Biochem. 234(2) (1995), 563; E. P. Jaeger, P. C. Jurs and T. R. Stouch, Eur. J. Med. Chem. 28(4) (1993), 275).
All retinoids have the same characteristic properties as highly UV-active chromophore and have low solubility in aqueous medium and are chemically unstable. This is why, in nature, retinoids bind to specific retinoid-binding proteins which confer protection, solubility and transportability in body fluids. A major problem in relation to the administration of vitamin A acid as pharmacological active substance is the need for immobilization. One possibility of achieving such immobilization and thus a protection of vitamin A acid is to bind it to a protein, as demonstrated in nature. A successful example of this strategy was shown by Zanotti et al., who cocrystallized transthyretin and vitamin A acid. This procedure is, however, difficult and cost-intensive.
European Patent 0 680 748 A1 discloses a composition in the form of a gel which contains an acidic, hydrophilic medium and at least one gel former which is formed from a crosslinked cationic polymer, which is characterized in that the hydrophilic medium is a medium which contains an amount of organic solvent which is 20 to 90% of the total weight of the composition, and contains an amount of water which is not more than 45% of the total weight of the composition, the gel former conferring on the composition a macroscopically homogeneous appearance of a gel and stability, and the cosmetic use of this gel, in particular for skin depigmentation. Gels of this type have an amorphous structure, and their viscosity and thus also their stability is determined by the degree of crosslinking of the polyelectrolytes. The release of a substance present in this gel in unbound form, such as, for example, retinoate, can be controlled by adjusting the viscosity of the gel. The high content of organic solvent in this gel is disadvantageous for use as medicinal product.
It was therefore the object of the present invention to provide a possibility for the immobilization of vitamin A acid which can be carried out easily and with maximal cost-efficiency.
This object is achieved according to the present invention by the provision of mesomorphic complexes of vitamin A acid and cationic polyelectrolytes.
The complexation of vitamin A acid with cationic polyelectrolytes is based on the finding that the formation of ordered structures in solution or in the solid state often takes place by means of self-organization by attachment of a surface-active agent to a polyelectrolyte. The driving force for this process are electrostatic and hydrophobic interactions in aqueous solution. A detailed investigation of self-organized complexes of synthetic polypeptides with surface-active agents having the opposite charge and a low molecular weight has recently been published by E. A. Ponomarenko, A. J. Waddon, D. A. Tirrell and W. J. MacKnight, Langmuir 12 (1996), 2169; A. Ponomarenko, A. J. Waddon, K. N. Bakeev, D. A. Tirrell and W. J. MacKnight, Macromolecules 29 (1996), 4340. It was additionally shown that the complexation of surface-active agents with polyelectrolytes results in a large number of stable mesophases of great structural diversity (M. Antonietti, J. Conrad and A. Thxc3xcnemann, Macromolecules 27 (1994), 6007; M. Antonietti, S. Henke and A. Thxc3xcnemann, Advanced Materials 8 (1996), 41; M. Antonietti, A. Kaul and A. Thxc3xcnemann, Langmuir 11 (1995), 2633). It has also been found that not only synthetic surface-active agents but also amphiphilic compounds might be suitable for this purpose. Vitamin A acid is, on the one hand, polar owing to the presence of the carboxyl functionality and, on the other hand, hydrophobic owing to the presence of the hydrophilic head group and the long hydrocarbon moiety (FIG. 1), that is to say an amphiphilic compound.
Three different polyelectrolytes are preferably used for the complexation of vitamin A acid for the purpose of the present invention. One which has been used is PDADMAC (poly (dimethyldiallylammonium chloride) which is known to form stable soluble complexes with natural lipids (M. Antonietti, A. Kaul and A. Thxc3xcnemann, Langmuir 11 (1995), 2633; M. Antonietti, A. Wenzel and A. Thxc3xcnemann, Langmuir 12 (1996), 2111) and forms gels with supramolecular ordering with sodium dodecyl sulfate (F. Yeh, E. L. Sokolov, A. R. Khokhlov and B. Chu, J. Am. Chem. Soc. 118 (1996), 6615). Hence a complex of vitamin A acid with PDADMAC is particularly preferred according to the invention.
Further particularly preferred, structurally different cationic polyelectrolytes which are particularly suitable for complexation for the purpose of the present invention are PM4VP, poly(N-methyl-4-vinyl-pyridine chloride), a polyelectrolyte with charges on the side groups (B. Philipp, W. Dawydoff and K. -J. Linow, Z. Chem. 22 (1982), 1) and poly(ionene-6,3) with the positive charges directly on the main polymer chain (FIG. 2), with PM4VP being referred to as a pendant type polyelectrolyte and poly(ionene-6,3) being called an integral type polyelectrolyte. In respect of its charges, PDADMAC occupies an intermediate position between PM4VP and poly(ionene-6,3). For this reason, PDADMAC is referred to as an intermediate type polyelectrolyte. It is also particularly preferred to use polyethyleneimine, obtainable from BASF, Ludwigshafen, Germany, which is marketed under the Lupasol trademarks.
Further polyelectrolytes which are particularly preferably used are poly-L-amino acids, in particular poly-L-arginine, poly-L-histidine, poly-L-lysine or a mixture thereof. The release behavior of the vitamin A acid present in the complex can be adjusted as required by the choice of the cationic polyelectrolyte.
The ratios of the vitamin A acid and the cationic polyelectrolyte in the complexes according to the invention may vary, with a ratio of 1:1 being particularly preferred. The complexes according to the invention can also easily be processed to film-like structures, so that they are in the form of a visco-elastic film, which have interesting physical properties. In contrast to relatively friable crystalline vitamin A acid, complexes with poly-electrolytes are highly deformable viscoelastic materials. These materials according to the invention show lamellar structures.
In a particularly preferred embodiment, the complexes according to the invention are in the form of particles in a nanodispersion together with a dispersing aid, the particle diameter being xe2x89xa65000 nm. All conventional dispersing aids known to the skilled person can be used in this nanodispersion according to the invention, with poloxamer 188 being preferred.
The ratios of the amounts of the complex and of the dispersing aid can be varied in order to obtain a nanodispersion with the properties required in each case, such as, for example, particle size, vitamin A acid release behavior etc. The ratio of complex to dispersing aid is preferably 1:10 to 10:1, particularly preferably 1:2 to 2:1, and it is most preferred for the complex and the dispersing aid to be present in equal amounts in the nanodispersion.
The particle diameter of the nanodispersion is selected appropriate for the requirements for the application. It is preferably 200 to 5000 nm, preferably 250 to 3000 nm, particularly preferably 300 to 2000 nm and most preferably 350 to 1500 nm. In another preferred embodiment the particle diameters are 350 to 400 nm, particularly preferably 350 to 390 nm and most preferably 1350 to 1460 nm. It has surprisingly emerged that the nanodispersion according to the invention is suitable not only for extracorporeal but also for intravenous applications.
The complexes according to the invention and, in particular, PDADMAC retinoate, poly(ionene-6,3) retinoate and PM4VP retinoate are soluble in a large number of polar organic solvents such as methanol, ethanol, 2-butanol, isopropanol and chloroform. Polyelectrolyte complexes with surface-active agents very probably dissociate at least partly in polar solvents (M. Antonietti, S. Fxc3x6rster, M. Zisenis and J. Conrad, Macromolecules 28 (1995), 2270), whereas such complexes may remain associated in solvents of low polarity (K. Bakeev, S. a. Chugunov, I. Teraoka, W. J. MacKnight, A. B. Zezin and V. A. Kabanov, Macromolecules 27 (1994), 3926). The solubility of the preferred complexes according to the invention is consistent with recently published investigations on complexes which consist of conventional synthetic poly-electrolytes and surface-active agents having the opposite charge (M. Antonietti, J. Conrad and A. Thxc3xcnemann, Macromolecules 27 (1994), 6007).
It has emerged that the complexes according to the invention are suprisingly mechanically stable without crosslinkers, and the stability of the complex according to the invention can be adjusted variably. This means that it is possible in an advantageous manner for the kinetics of release of vitamin A acid from the complex to be controlled in a specific manner and adapted to the particular requirements for application. It has proved to be particularly beneficial that the complexes are mesomorphic with a lamellar structure and, in a particularly preferred embodiment, a physical order state which corresponds to that of a smectic liquid crystal exists.
The complexes according to the invention have advantages in particular by comparison with gels known from the prior art, which are amorphous and for which the chemical behavior of the substances present in them, in particular their release behavior, is determined by the degree of crosslinking, such as, for example, cost-effective production, good storability, simple processibility and usability etc.
In addition, the complexes according to the invention contain only small amounts of or absolutely no organic solvents, so that it has been possible to avoid the use, which is increasingly regarded as critical, of organic solvents, in particular for pharmaceutical applications.
The present invention further relates to a process for the preparation of the complexes according to the invention of vitamin A acid and cationic polyelectrolytes, in which solutions of vitamin A acid and of a polyelectrolyte are mixed, and the crude complexes which have formed are isolated and, where appropriate, purified by methods known per se. In a preferred embodiment of the invention, the polyelectrolyte used is PDADMAC, PM4VP, poly(ionene-6,3), polyethyleneimine or poly-L-amino acids, in particular poly-L-arginine, poly-L-histidine, poly-L-lysine or a mixture thereof. In another preferred embodiment, the process according to the invention is carried out in basic solution, preferably by dissolving vitamin A acid in a basic aqueous solution and then adding an aqueous solution of the polyelectrolyte, preferably dropwise. The complexes according to the invention precipitate during the addition and can easily be removed. For example, further purification can take place by redissolving in methanol, and excess vitamin A acid and salt can be removed by ultrafiltration. The process according to the invention is normally carried out at room temperature, preferably at 20xc2x0 C. to 30xc2x0 C., but at not more than 60xc2x0 C. to 80xc2x0 C., particularly preferably xe2x89xa630xc2x0 C.
The present invention further relates to the use of the mesomorphic complexes according to the invention, in particular in the form of viscoelastic films or nanodispersions, as vitamin A substitute. It is now possible to use the mesomorphic complexes, which are preferably in the form of viscoelastic films which contain immobilized vitamin A acid, particularly preferably in the form of a nanodispersion, in place of the pure acid for all uses of vitamin A acid for which, in particular, the instability of the acid was disadvantageous.
The complexes according to the invention, in particular in the form of films or nanodispersions, are particularly preferably used as active pharmaceutical ingredients, a suitable and preferred area of application being at present in particular skin disorders or inhibition of the growth of malignant tumors.
The present invention therefore further relates to pharmaceutical compositions which contain the mesomorphic complexes according to the invention, in particular in the form of viscoelastic films or nanodispersions, of the present invention. Pharmaceutical compositions of this type can be employed wherever vitamin A acid or other retinoids have been employed to date.
The complexation of vitamin A acid with cationic polyelectrolytes of various structures (for example integral, intermediate and pendant type) results in the formation of novel materials according to the invention with interesting structural and optical properties as well as novel pharmaceutical compositions. Their main properties are:
1. The novel mesomorphic complexes contain up to 70% by weight optically active molecules. Because of the strong chromophoric interactions in the solid state, the complexes show an additional strong high-energy absorption at 252 nm. In addition, the solid phase Uv/vis spectrum can be significantly influenced by additional chromophores such as, for example, methyl-4-vinylpyridine, which provides further possibilities for altering the absorption characteristics.
2. The complexes can easily be processed to nanodispersions or to films with diverse lamellar structures, which show great morphological similarity to SA liquid crystals.
3. Depending on the polyelectrolyte structure, the glass transition temperature can be adjusted in the range between xe2x88x9219 and 28xc2x0 C., and the mechanical properties are also variable within a wide range. From the pharmaceutical viewpoint, the complexes can be regarded as novel formulation of a very active substance. It is to be assumed that the complexes have a reduced toxicity and a reduced teratogenic effect compared with conventional formulations containing vitamin A acid. The complexes can be used to treat skin disorders such as, for example, acne, psoriasis and hyperkeratoses. The formulation of the complexes as colloidal particles might be another way of utilizing the pharmaceutical potential of vitamin A acid for example as active substance for inhibiting the growth of malignant tumors. Vitamin A acid bound ionically to various polyelectrolytes is moreover a promising material for biomimetic applications. It can be assumed that the complexes can also be used as part of a photosynthetic system, in which case protons are transported from the inside of a membrane to the outside, and thus there is formation of an electrochemical gradient which presumably promotes ATP synthesis. In any event, the optical activity of the natural photosensitive pigments is of considerable interest because it allows conclusions to be drawn both about the protein-chromophore interaction and about conformational changes occurring after absorption of light. It is to be assumed that investigation of the uniaxially aligned multilamellar complex films will allow the understanding of the molecular basis of the optical activity of complexes in natural systems to be advanced.
A further embodiment relates to a method for treating a patient with the complexes according to the invention, which are in the form, in particular, of a film or nanodispersion, who is suffering from skin disorders, in particular acne, psoriasis or hyperkeratoses, or from malignant tumors.